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On some toxinological aspects of the starfish Stellaster equestris (RETZIUS,

1805)

Article  in  Journal of Venomous Animals and Toxins including Tropical Diseases · January 2008
DOI: 10.1590/S1678-91992008000300005

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Received: October 16, 2007 J. Venom. Anim. Toxins incl. Trop. Dis.
Accepted: April 23, 2008 V.14, n.3, p. 435-449, 2008.
Abstract published online: May 12, 2008 Original paper.
Full paper published online: August 31, 2008 ISSN 1678-9199.

ON SOME TOXINOLOGICAL ASPECTS OF THE

STARFISH Stellaster equestris (RETZIUS, 1805)

KANAGARAJAN U (1), BRAGADEESWARAN S (1), VENKATESHVARAN K (2)

(1) Centre of Advanced Study in Marine Biology, Parangipettai, Tamil Nadu, India; (2)
Aquatic Biotoxinology Laboratory, Central Institute of Fisheries Education, Mumbai,
Maharashtra, India.

ABSTRACT: Whole-body extracts in methanol were obtained from the starfish

Stellaster equestris. The crude toxin was fractionated stepwise using
diethylaminoethyl (DEAE) cellulose column chromatography. The crude toxin was
lethal to male albino mice at a dose of 1.00 mL (containing 531.0 µg/mL protein)
when injected intraperitoneally (IP) but the toxicity was abolished in all cases except
one upon fractionation. The crude toxin and all the adsorbed fractions exhibited
potent hemolytic activity on chicken, goat and human blood. However, group B
human erythrocytes were resistant to lysis by all fractions and group O by most of the
fractions. Paw edema in mice was caused by the crude toxin and all fractions.
Pheniramine maleate and piroxicam blocked the toxicity when administered earlier
than, or along with, the crude or fractionated toxins but not when administered after
the envenomation. Pretreatment with either of these drugs also blocked edema
formation.

KEY WORDS: starfish, toxicity, hemolysis, human blood groups, paw edema.

CONFLICTS OF INTEREST: There is no conflict.

FINANCIAL SOURCE: Tamil Nadu State Council of Science & Technology, Chennai,
India.

CORRESPONDENCE TO:
K. VENKATESHVARAN, Aquatic Biotoxinology Laboratory, Central Institute of
Fisheries Education, Seven Bungalows, Versova, Mumbai 400 061, India. Fax: +91
22 2636 1573. Email: [email protected], [email protected].
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 436

INTRODUCTION
The phylum Echinodermata includes a diverse group of typically slow-moving and
non-aggressive marine animals, including three venomous classes, namely the
Asteroidea (starfishes), Echinoidea (sea urchins), and Holothuroidea (sea
cucumbers). The crown-of-thorns starfish, Acanthaster planci, being venomous,
accounts for the majority of human envenomations (27) but poisonous starfish also
affect health in countries including Taiwan where they are consumed (15, 42). A
variety of toxins, which are comparable with the most toxic organophosphate
chemical warfare agents (28), are reported from starfish; these include saponins (11),
asterosaponins (sulfated steroidal glycosides) (40), free and sulfated sterols (17, 18,
19), and protein-like substances (39), besides tetrodotoxin (TTX) (20, 41) and
paralytic shellfish poison (PSP) (2, 21). However, these toxins can also be used as
guides leading to new, more effective therapies, because their targets of action are
well known (9, 28). Despite the occurrence of around 350 species under 103 genera
in Indian waters, echinoderms have been poorly studied in this country especially
with reference to their toxicity and pharmacological potential. Hence, the present
study focused on the biotoxinological aspects of the starfish Stellaster equestris from
Cuddalore on the eastern seaboard of India.

MATERIALS AND METHODS

Specimen Collection
The starfish Stellaster equestris (Retzius, 1805) (Echinodermata: Asteroidea:
Valvatida: Goniasteridae) was collected from the Fishing Harbor at Cuddalore
(11°45’N, 79°47’E) and Mudasalodai Fish Landing Center at Parangipettai (11°32’N,
79°45’E), brought fresh to the laboratory (32 km and 2 km, respectively) and
immediately air dried for subsequent use.

Preparation of Crude Toxin

Crude toxin was prepared according to previous works with certain modifications (8).
Samples were air dried for ten days and, after complete drying, were fully immersed
in methanol, covered and maintained for five days. The starfish were then removed
after squeezing them, and the solvent was filtered through Whatman® filter paper n.
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 437

1 (0.4 µm); it was then evaporated at low pressure using an R-200 Büchi Rotavapor®
at 30°C. The resultant compound was stored at 4°C for further use as crude toxin.

Partial Purification Using Column Chromatography

The crude toxin was dissolved in phosphate buffer (pH 7.4) at 5 mg/mL and eluted
through a DEAE cellulose column (height 32 cm; diameter 2 cm). Five unadsorbed
fractions, 15 mL each, were eluted with phosphate buffer; and were later pooled and
referred to as fraction UA. Ten adsorbed fractions, each 15 mL, were eluted with a
step gradient of 0.1 to 1.0 M NaCl in phosphate buffer, and designated as fractions
F1 to F10. All fractions were stored at 4°C for further use.

Protein Estimation
Crude protein content and partially purified fractions were estimated according to
previous works (30) using bovine serum albumin (BSA) at the rate of 1 mg/1 mL as
the standard.

Mice Bioassay for Lethality

All animal bioassays were carried out following the statement of the Institute Ethical
Committee. The bioassay for lethality was done using clinically healthy male albino
mice weighting 20 ± 2 g that were maintained in a healthy condition in the laboratory.
Mice in triplicate sets were challenged intraperitoneally with 0.25, 0.50, 0.75 and 1.0
mL of the crude toxin, dissolved at 5 mg/mL in phosphate buffer saline (PBS); and
with 1.0 mL each of the 11 fractions. A control was maintained in each case by
injecting an equal volume of PBS (pH 7.4). The times of injection and death, besides
behavioral changes before death, were recorded. Mice that died upon envenomation
were autopsied to observe gross anatomical alterations.

Tests for Stability of the Crude Toxin

Tests were conducted in accord with previous works to assess the stability of the
crude toxin upon (35):
• heating at 50°C, 60°C, 80°C and 100°C in a water bath for five minutes;
• autoclaving at 120°C for 15 minutes;
• changing of pH (3.0 – 8.0); and
• storing at –20°C for more than one year (13 months).
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 438

Hemolytic Activity
The crude toxin and the 11 fractions were assayed for their hemolytic activity on
chicken, goat and human (A, B, AB and O) erythrocytes.
Samples of chicken and goat blood were obtained from a nearby slaughterhouse,
while the human blood was obtained from Sheena Clinic, Andheri (W), Mumbai,
using 2.7% ethylenediaminetetraacetic acid (EDTA) solution as an anticoagulant at
5% of the blood volume, and brought to the laboratory. The blood was centrifuged
thrice at 5,000 rpm for five minutes; a 1% erythrocyte suspension was prepared by
adding 99 mL normal saline to 1 mL of packed erythrocytes.
The microhemolytic test was performed in 96-well ‘v’ bottom microtitre plates. Serial
two-fold dilutions of the crude toxin/fractions were made in 100 µL of normal saline.
Then 100 µL of 1% erythrocyte was added to all the wells. For positive control, 100
µL of distilled water, and for negative control 100 µL of normal saline were added
respectively to the 1% red blood cell (RBC) suspension. The plate was gently shaken
and allowed to stand for two hours at room temperature. Presentation of uniform red-
color suspension in the wells was considered to be positive hemolysis and a button
formation in the bottom of the wells constituted a lack of hemolysis. The reciprocal of
the highest dilution of the crude toxin showing the hemolytic pattern (hemolytic unit)
was divided by the protein content to obtain the specific hemolytic unit.

Edema Formation
Following previous works (37), a group of two mice in each case was injected
subplantarly with 0.1 µL of the crude toxin/fraction in the right footpad and with 0.1
mL of buffered saline in the left footpad. Two hours after injection, percentage of size
increase was measured with a Vernier caliper and the growth of the envenomated
paw relative to the saline-injected paw was taken as the edema ratio (ER). The
minimum edematous dose was defined as the dose causing 105% ER.

Antidote Experiments
Commercially available antihistaminic drug, pheniramine maleate (Avil®, Hoechst),
and an antiprostaglandin analgesic, piroxicam (Dolonex®, Pfizer) in injectable form,
were procured locally from qualified pharmacists. Each of the drugs was used at a
concentration of 0.2 mg/20 g body weight of mice; this dose was deduced based on
the dose prescribed by the manufacturers for human use, based on body weight.
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 439

These two medications were selected since they are commonly used in India for the
symptomatic relief of most envenomations in humans, though there are no recorded
starfish poisonings or envenomations from this country. The antidote investigations
were made using the crude toxin and those fractions which were lethal to mice.
Control was maintained by injecting toxins (IP) at the previously determined lethal
dose for each toxin but without any antidote. All experiments were carried out in
triplicate sets.
The injectable solution was diluted further with PBS to obtain sufficient injectable
volume for a dose as low as 0.2 mg. Three types of blocking experiments –
pretreatment, cotreatment and post-treatment – were carried out:
• Pretreatment model: the test mice were injected IP with 0.9 mL (containing 0.2
mg of the active component) of pheniramine maleate/piroxicam, and set free
in the cage. After 30 minutes, the animals were injected IP with the
predetermined lethal dose, and kept under observation for 24 hours.
• Cotreatment model: 0.9 mL of pheniramine maleate/piroxicam and the
predetermined lethal dose of the toxin were taken together in one syringe and
injected IP to mice, which were then placed under observation for 24 hours.
• Post-treatment model: the mice were first injected with the predetermined
lethal dose of the toxin. Immediately after the appearance of envenomation
symptoms of, 0.9 mL of pheniramine maleate/piroxicam was administered IP
to the mice, after which they were observed for 24 hours.

Edema Inhibition Tests

Only the pretreatment model was used in this case; 1 hour after IP administration of
pheniramine maleate and piroxicam, at 0.2 mg/20 g body weight, the assay for paw
edema was conducted as described above. Control experiments were performed on
mice without the administration of drugs.

RESULTS
Crude Toxin
On an average, 5 mg of crude toxin was obtained from 50 g dry weight of the starfish.
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 440

Protein Content of the Crude Toxin/Fractions (Table 1)

Protein content in the crude toxin was 531 µg/mL while the amount of protein in the
purified fractions varied between 29 µg/mL (fractions F1, F5 and F7) and 68 µg/mL
(fraction F4).

Table 1. Protein content of crude toxin, and its partially purified fractions, from the
starfish Stellaster equestris (all values are means of triplicate sets)

Sample Tested Amount of protein (µg/mL)

Crude toxin 531.0 ± 2.3
F1 29.6 ± 0.8
F2 39.1 ± 1.2
F3 53.1 ± 1.4
F4 68.8 ± 0.9
F5 28.0 ± 0.7
F6 41.0 ± 1.0
F7 21.9 ± 0.5
F8 53.0 ± 0.9
F9 65.5 ± 1.1
F10 48.9 ± 0.8
UA Non-detectable

Toxicity in Mice Models

Both crude toxin at a dose of 1.0 mL (containing 5 mg of crude toxin) and fraction F9
at a dose of 1 mL (containing 65.5 µg/mL) protein were lethal to mice upon IP
injection; however, in all cases, various toxicity symptoms were observed (Table 2).
 U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 441

Table 2. Toxicity of starfish crude toxin and its fractions (at 5.0 mg/mL) IP in male
albino mice (20 ± 2 g)

Serial Extract/ Injected Death time

Symptoms Autopsy
number fractions volume (mL) (min:s)
Dark discoloration
Palpitation, escape reaction,
Crude toxin of liver,fluid
1 1.00 7:50 dragging of hind limbs,
accumulation in
paralysis, and coma – lethal
visceral cavity
Crude toxin Palpitation, excess defecation –
2 0.75 – –
not lethal
Crude toxin Palpitation, excess defecation –
3 0.50 – –
Not lethal
Crude toxin Palpitation, excess defecation –
4 0.25 – –
not lethal
Palpitation, escape reaction –
5 UA 1.00 – –
not lethal
Palpitation, micturition, escape
6 F1 1.00 – –
reaction – not Lethal
Palpitation, escape reaction –
7 F2 1.00 – –
not lethal
8 F3 1.00 – Palpitation – not lethal –
Palpitation, escape reaction –
9 F4 1.00 – –
not lethal
No symptoms –
10 F5 1.00 – –
not lethal
Palpitation, micturition – not
11 F6 1.00 – –
lethal
12 F7 1.00 – Palpitation – not lethal –

Palpitation, escape reaction,

13 F8 1.00 – –
excess defecation – not lethal
Granular
appearance and
dark discoloration
Palpitation, micturition, sudden
14 F9 1.00 0:46 of kidney, non-
death – lethal
specific
hemorrhage in
viscera
Palpitation, sniffing, escape
15 F10 1.00 – –
reaction – not lethal
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 442

Stability Tests
Lethal activity of the crude extract was not affected after storage for 12 months at
20°C. The crude extracts were found to be thermolabile above 60°C. The crude toxin
lost its activity in acidic pH; mice injected with it showed some symptoms of toxicity
initially, but recovered within a few minutes.

Hemolytic Assay
The crude toxin as well as the fractions produced pronounced hemolytic activity on
chicken, goat and human erythrocytes (Figure 1). Hemolytic factors were present in
the crude toxin as well as in all the fractions except UA, but differed considerably
depending on the type of blood used. Chicken blood, like groups A and AB of human
blood, were the most vulnerable to lysis provoked by the starfish extracts. None of
the fractions could lyse group B erythrocytes while group O erythrocytes were lysed
by only two out of the ten fractions.

Figure 1. The extent of in vitro hemolytic activity of Stellaster equestris crude toxin
(fractions F1 to F10) on chicken, goat and human blood.
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 443

Antidote Investigation
Both the antidotes tested – pheniramine maleate and piroxicam – negated the toxicity
of the crude toxin when injected prior to envenomation or together with the toxin.
Injecting each the drugs after envenomation, could only delay the onset of death and
were not sufficiently effective to prevent mortality.

Edema Formation
Mice paw edema was caused invariably by the crude toxin and all the fractions
(Figure 2) with ER between 157 and 334%. Neither of the drugs tested could block
the edematous activity of fractions F1, F2, F8, F9, and UA but was able to variably
reduce the ER in all other cases, sometimes even below the stipulated 105%.

Figure 2. The extent of paw edema formation in male albino mice (20 ± 2 g) provoked
by Stellaster equestris crude toxin (fractions F1 to F10) and the blocking of
edematous activity by piroxicam and pheniramine maleate.
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 444

DISCUSSION
Methanol is considered as a universal solvent which could extract even the basic
proteins without exempting any one of them; methanolic extracts of the whole body of
the starfish Anthopleura fuscoviridis yielded two toxins AFT-I and AFT-II, that were
lethal to mice (38). The protein levels observed during the present study compares
well with previous results (36) that reported protein values as high as 4,820 mg/227 g
of tissue from Acanthaster planci.
Our data further support the earlier findings that crude extracts of some echinoderms
(4, 5, 24, 33, 43) and others extracted from spines of Acanthaster planci were lethal
to mice (39). Sulfated saponins of echinoderms are toxic to fishes, annelids,
arthropods etc. (10). The symptoms observed in envenomated mice in our study are
indicative of central nervous system (CNS) toxicity and nephrotoxicity, concomitant
with those reported for asterosaponins and saponins. Asterosaponins provoked
paralysis in mice (34) while toxins of the British starfish, Marthasterias glacialis
caused convulsions and drowsiness followed by collapse and death in mice (23).
Our data suggest that the lethal element in the present crude toxin is different from
those factors exhibiting hemolytic action, as had also been observed in the case of
Acanthaster planci (39).
Metabolites from echinoderms tend to cause cell lysis and hemolysis (29, 31, 32), as
found in the present study. A partially purified product from the starfish Asterias
amurensis was hemolytic to rabbit red blood cells, toxic to killifish, lethal to fly
maggots and earthworms, and emetic to cats (43). Five of the nine polar steroids
isolated from the alcoholic extract of the Far Eastern starfish Henricia leviuscula
showed moderate hemolytic activity in the mouse erythrocyte assay (16).
It is very interesting that within the ABO system of human blood, group B resisted
lysis by all the fractions while group O resisted lysis by most of them. Despite our
best efforts we could not locate any previously published data on such an effect by
invertebrate or vertebrate toxins. But considerable literature exists on this effect in
the case of bacterial toxins. It has been shown that individuals with type O blood are
more susceptible than other individuals to severe cholera (14). Compared to
community controls in Bangladesh, cholera patients were twice as likely to have
blood group O and one-ninth as likely to present blood group AB (12).
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 445

Similarly, it has been shown that the differential interaction of Escherichia coli, heat-
labile toxin like cholera toxin with intestinal brush border glycoproteins of pigs (7) and
rabbits (6) are dependent upon ABH and related blood group antigenic determinants.
Our data assume significance and indicate that further in vitro studies are essential in
order to clarity possible inter-individual (and possibly inter-regional/inter-racial)
variability in susceptibility to the toxic effects of various plant and animal poisons and
venoms.
Present results on the toxin stability corroborate those pertaining to A. planci (39);
this toxin was non-dialyzable, and it lost its activity at 60°C or at pH lower than 3.0 or
higher than 10.0. In addition the toxin that was exposed to repetitive freezing (at –
20°C) and thawing was inactivated. From these observations, it was considered that
A. planci toxin was probably a protein-like substance (39).
Similar instances of stability, as in the present study, have been encountered by
other researchers (25). The presently extracted toxins are comparatively
thermostable up to 60°C, possibly due to the tropical occurrence of the starfish from
which they were extracted, as had been suggested in the case of cytolysin from
Radianthus sp (26). The phospholipase A2 component in starfish toxin is said to be
unstable at 40°C and has been reported to lose its activity completely when
maintained for 25 minutes at 56°C (13). Susceptibility of the toxin to acidic pH
observed is in accordance with earlier reports (22).
A number of thermolabile biotoxins of echinoderms are known to cause edema in
experimental animal models and also in humans (37). Pretreatment and also
cotreatment of both antihistaminic (pheniramine maleate) and antiprostaglandin
analgesic (piroxicam) drugs have been effective in negating the toxicity of the crude
toxin like that of the lethal fractions of the starfish species. However, on post-
treatment, these compounds could only delay the onset of death, thus indicating that
their use is limited only to symptomatic relief. Action of catfish mucus toxin was
inhibited by pretreatment with atropine and indomethacin (1) while promoxine with
hydrocorticosone, prednisone and 1% prednisone acetate with hyocine 0.25% were
effective against nematocyst stings in humans (3).
Observed toxicity of the extracts may have been mediated by a prostaglandin
mechanism since piroxicam has been reported (1) to act by through blocking the
cyclooxygenase activity of prostaglandin synthesis. Also, in the present study,
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 446

pheniramine maleate, the antihistaminic drug, has either delayed or prevented death,
indicating the involvement of histamine or histamine-like compounds in the toxins that
caused death of test animals.
Between the two drugs tested, pheniramine maleate appears to be more effective
than piroxicam in blocking both the lethal effect and edema formation.

ACKNOWLEDGEMENTS
Thanks are due to the Director, Center of Advanced Study in Marine Biology,
Parangipettai, India and the Director, Central Institute of Fisheries Education,
Mumbai, India for facilities provided. The first author thanks the Tamil Nadu Council
of Science and Technology, Chennai, India for financial assistance.

REFERENCES
1 AL-HASSAN JM., THOMSON M., SUMMERS B., CRIDDLE RS., Purification and
properties of a hemagglutination factor from the Arabian Gulf catfish (Arius
thalassinus) epidermal secretion. Comp. Biochem. Physiol., 1986, 85B, 31-9.
2 ASAKAWA M., NISHIMURA F., MIYAZAWA K., NOGUCHI, T. Occurrence of
paralytic shellfish poison in the starfish, Asterias amurenis in Kure Bay, Hiroshima
Prefecture, Japan. Toxicon, 1997, 35, 1081-7.
3 AUERBACH, PS. Clinical therapy of marine envenomation and poisoning. In: Tu
AT (ed.) Handbook of Natural Toxins. Vol 3: Marine Toxins and Venoms. New York:
Marcel Dekker, 1988, 493-565.
4 BAKUS GJ. Chemical defenses mechanisms and fish feeding behaviour on the
Great Barrier Reef, Australia. Science, 1981, 211, 497-9.
5 BAKUS GJ., GREEN G. Toxicity in sponges and holothurians: a geographic
pattern. Science, 1974, 185, 951-3.
6 BALANZINO LE., BARRA JL., GALVÁIN EM., ROTH GA., MONFERRAN CG.
Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with
glycoconjugates from rabbit intestinal brush border membranes: relationship with
ABH blood group determinants. Mol. Cell. Biochem., 1999, 194, 53-62.
7 BALANZINO LE., BARRA JL., MONFERRAN CG., CUMAR FA. Differential
interaction of Escherichia coli heat-labile toxin and cholera toxin with pig intestinal
brush border glycoproteins depending on their ABH and related blood group
antigenic determinants. Infect. Immun., 1994, 62, 1460-4.
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 447

8 BRAEKMAN JC., DALOZE D., STOLLER C., VAN SOEST RWM. Chemotaxonomy
of Agelas (Porifera: Demospongiae). Bioch.l Syst. Ecol., 1992, 20, 417-31.
9 CATTERALL WA. Molecular properties of voltage-sensitive sodium channels.
Annu. Rev. Biochem., 1986, 55, 953- 85.
10 DHAWAN BN., GARG HS., GOEL AK., SRIMAL RC., SRIVASTAVA MN.,
BHAKUNI S. Bioactivity of marine organisms, part 7: screening of some marine fauna
from the Indian coasts. Indian J. Exp. Biol., 1993, 31, 505-10.
11 FINDLAY JA., JASEJA M., BURNELL DJ., BRISSON JR. Major saponins from the
starfish Asteria forbesi. Complete structures by nuclear magnetic resonance
methods. Can. J. Chem. 1987, 65, 1384 -91.
12 GLASS RI., HOLMGREN J., HALEY CE., KHAN MR., SVENNERHOLM A.,
STOLL BJ., HOSSAIN KMB, BLACK RE, M. YUNUS M., BARUA D. Predisposition
for cholera of individuals with O blood group: possible evolutionary significance. Am.
J. Epidemiol., 1985, 121, 791-6
13 GROTENDORST GR., HESSINGER DA. Enzymatic characterization of the major
phospholipase A2 component of sea anemone (Aiptasia pallida) nematocyst venom.
Toxicon, 2000, 38, 931-43.
14 HARRIS JB., KHAN AI., LAROCQUE RC., DORER DJ., CHOWDHURY F.,
FARUQUE AS., SACK DA., RYAN ET., QADRI F., CALDERWOOD SB. Blood group,
immunity, and risk of infection with Vibrio cholerae in an area of endemicity. Infect.
Immun., 2005, 73, 7422-7.
15 HWANG DF. Research on marine toxins in Taiwan. J. Toxicol. Toxin Rev., 2003,
22, 663-78.
16 IVANCHINA NV., KICHA AA., KALINOVSKY AI., DMITRENOK PS.,
DMITRENOK AS., CHAIKINA EL., STONIK VA., GAVAGNIN M., CIMINO G. Polar
steroidal compounds from the far eastern starfish Henricia leviuscula. J. Nat. Prod.,
2006, 69, 224-8.
17 IVANCHINA NV., KICHA AA., KALINOVSKY AI., DMITRENOK PS., STONIK VA.,
Hemolytic steroid disulfates from the far eastern starfish Pteraster pulvillus J. Nat.
Prod., 2003, 66, 298-301.
18 IVANCHINA NV., KICHA AA., KALINOVSKY AI., DMITRENOK PS., STONIK VA.,
RIGUERA R., JIMENEZ C. Hemolytic polar steroidal constituents of the starfish
Aphelasterias japonica. J. Nat. Prod., 2000, 63, 1178-81.
U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 448

19 KAPUSTINA II., PONOMARENKO LP., MOISEENKO OP., STONIK VA. Free and
sulfated sterols of two far-east leptasterias starfish. Chem. Nat. Comp., 2001, 37,
515-9.
20 LIN SJ., HWANG DF. Possible source of tetrodotoxin in the starfish Astropecten
scoparius. Toxicon, 2001, 39, 573-9.
21 LIN SJ., TSAI YH., LIN HP., HWANG DF. Paralytic toxins in Taiwanese starfish
Astropecten scoparius. Toxicon, 1998, 36, 799-803.
22 MACEK P., LEBEZ D. Isolation and characterization of three lethal and hemolytic
toxins from the sea anemone Actinia equina L. Toxicon, 1988, 26, 441-51.
23 MACKIE AM., LASKER R., GRANT PT. Avoidance reactions of a mollusc
Buccinum undatum to saponin-like surface-active substances in extracts of starfish
Asterias rubens and Marthasterias glacialis. Comp. Biochem. Physiol., 1968, 26,
415-28.
24 MACKIE AM., SINGH HT., FLETCHER TC. Studies on the cytolytic effects of
seastar (Marthasterias glacialis) saponins and synthetic surfactants in the plaice
Pleuronectes platessa. Mar. Biol., 1975, 29, 307-14.
25 MAHNIR VM., KOZLOVSKAYA EP., KALINOVSKY AI. Sea anemone Radianthus
macrodactylus - a new source of palytoxin. Toxicon, 1992, 30, 1449-56.
26 MONASTYRNAYA MM., ZYKOVA TA., APALIKOVA OV., SHWETS TV.,
KOZLOVSKAYA EP. Biologically active polypeptides from the tropical sea anemone
Radianthus macrodactylus. Toxicon, 2002, 40, 1197-217.
27 MONICO EP., CALISE A., NOTTINGHAM D. Marine envenomations among home
aquarium hobbyists. Internet J. Emer. Inten. Med. 2007, 10.
28 MUNRO NB., AMBROSE KR., WATSON AP. Toxicity of the organophosphate
chemical warfare agents GA, GB and VX: Implications for public protection. Environ.
Health Perspect., 1994, 102, 18 - 37.
29 NAIK CG., KAMAT SY., PARAMESWARAN PS., DAS B., PATEL J., RAMANI P.,
BHAKUNI DS., GOEL AK., JAIN S., SRIMAL RC. Bioactivity of marine organisms.
Part 5. Screening of some marine animals from the Indian coast. Mahasagar, 1989,
22, 99-104.
30 PETERSON GL. A simplification of the protein assay method of Lowry et al. which
is more generally applicable. Anal. Biochem., 1977, 83, 346-56.
31 RAO DS., JAMES DB., GIRIJAVALLABHAN KG., MUTHUSWAMY S.,
NAJMUDDIN M. Biotoxicity in echinoderms. J. Mar. Biol. Ass. India, 1985, 27, 88-96.
 U. Kanagarajan et al. ON SOME TOXINOLOGICAL ASPECTS OF THE STARFISH Stellaster equestris
(RETZIUS, 1805). J. Venom. Anim. Toxins incl. Trop. Dis., 2008, 14, 3, p. 449

32 RAO DS., JAMES DB., GOPINATHAPILLAI CS., THOMAS PA., APPUKUTTAN

KK., GIRIJAVALLABHAN KG., GOPINATHAN CP., MUTHUSWAMY S., NAMUDDIN
M. Biotoxicity in Marine Organisms. In: Bioactive Compounds from Marine Organisms
. Proceedings of an Indo – US Symposium (edited by Marg – Frances Thompson, R.
Sarojini and R. Nagabhushanam), New Delhi: Oxford and IBH Publishing Co. Pvt.
Ltd., 1991, 367-71.
33 RICKLEFS, RE. Ecology. 2.ed. New York: Chiron Press, 1979, 966p.
34 SANCHEZ J., BRUHN T., ANEIROS. A, WACHTER E., BERESS L. A Simple
biochemical method in the search for bioactive polypeptides in a sea anemona
(Anemonia sulcata). Toxicon, 1996, 34, 1361-6.
35 SHIOMI K., QIAN WH., LIN XY., SHIMAKURA K., NAGASHIMA Y., ISHIDA M.
Novel polypeptide toxins with crab lethality from the sea anemone Anemonia
erythraea. Biochem. Biophys. Acta, 1997, 1335, 191-8.
36 SHIOMI K., YAMAMOTO S., YAMANAKA H., KIKUCHI T. Purification and
characterization of a lethal factor in venom from the crown-of-thorns starfish
(Acanthaster planci). Toxicon, 1988, 26, 1077-83.
37 SMITH M. Skin problems from marine echinoderms. Dermatologic Therapy, 2002,
15, 30-3.
38 SUNAHARA S., MURAMOTO K., TENMA K., KAMIYA H. Amino acid sequence of
two sea anemone toxins from Anthopleura fuscoviridis. Toxicon, 1987, 25, 21-19.
39 TAIRA E., TANAHARA N., FUNATSU M. Studies on the toxin in the spines of
starfish Acanthaster planci. I. Isolation and some properties of the toxin found in
spines. Sci. Bull. Coll. Agri. Univ. Ryukyus, 1975, 22, 203-13.
40 TANG HF., YI YH., LI L., SUN P., ZHANG SQ., ZHAO YP. Three new
asterosaponins from the starfish Culcita novaeguineae and their bioactivity. Planta
Med. 2005, 71, 458-63.
41 TSAI YH., CHAO SM., LIN GT., NOGUCHI T., HWANG DF. Tetrodotoxins of the
starfish Astropecten vappa collected from western Taiwan. Fisher. Sci., 2004, 70,
930-2.
42 US FOOD AND DRUG ADMINISTRATION. Foodborne Pathogenic
Microorganisms and Natural Toxins Handbook. Tetrodotoxin.
http://www.cfsan.fda.gov/~mow/chap39.html [Accessed 25 April 2006].
43 YASUMOTO T., TANAKA M., HASHIMOTO Y. Distribution saponins in
echinoderms. Bull. Jap. Soc. Sci. Fish., 1966, 32, 673-6.

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